Molecular characterization of a Brucella species large DNA fragment deleted in Brucella abortus strains: evidence for a locus involved in the synthesis of a polysaccharide.

A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced B. melitensis DNA fragment.     

Characterization of smooth Brucella lipopolysaccharides and polysaccharides by monoclonal antibodies.

Two mouse monoclonal antibodies (mAb) generated to the M antigen of Brucella melitensis 16M were analysed. Binding profiles of both monoclonals were established by a competitive enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides, O polysaccharides and native hapten polysaccharides from B. melitensis 16M, B. abortus 544 and Yersinia enterocolitica O:9. Using this assay, significant differences in the reactivity of both antibodies were found with A and M antigens from Brucella spp. and the O polysaccharide from Y. enterocolitica O:9. These findings are consistent with the simultaneous expression of A and M epitopes on the lipopolysaccharide of all smooth Brucella strains. Quantitatively similar inhibitory powers were established for the native hapten and O polysaccharide from B. melitensis 16M. However, different behaviour was observed between both antigenic preparations obtained from B. abortus 544. The use of lipopolysaccharide-M-specific mAb in different serological tests instead of polyclonal antisera may be of great practical use for minimizing the risk of the appearance of cross-serological reactions between smooth Brucella strains and Y. enterocolitica O:9.     

Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis

Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.     

Characterization of a native polysaccharide hapten from Brucella melitensis.

The 13C nuclear magnetic resonance spectrum of Brucella melitensis native polysaccharide hapten proved to be very similar to that generated by the O-specific chain (PS) isolated from B. melitensis lipopolysaccharide; that is, to a linear polymer in which the repeating unit is composed of five N-formylperosaminyl residues, one of them being substituted at position C-3 and the other four at position C-2. The serological analysis suggests that the so-called A determinant is present solely in Brucella abortus PS, the M determinant is only in B. melitensis PS, and the extensive cross-reaction observed is due to a determinant shared by both polysaccharides.     

Comparative analysis of Brucella serotype A and M and Yersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats.

Hapten polysaccharides of Brucella smooth M and A serotypes were prepared from Brucella sp. and Yersinia enterocolitica O:9 by previously described hydrolytic (O chain) or nonhydrolytic (native hapten [NH]) procedures. The purified polysaccharides differed only in the presence (O chain) or absence (NH) of lipopolysaccharide core sugars. The polysaccharides were compared by reverse radial immunodiffusion for the diagnosis of brucellosis in cattle (Brucella abortus biotype 1 [A serotype] and Brucella melitensis biotype 3 [AM serotype]), sheep (B. melitensis biotypes 1 [M serotype] and 3), and goats (B. melitensis biotype 1). The reverse radial immunodiffusion test with the NH from B. melitensis 16 M (serotype M) showed the highest sensitivity (89.6 to 97.3%), regardless of the host species and the serotype of the infecting Brucella sp. Y. enterocolitica O:9 NH (A serotype) was useful for diagnosing disease in cattle infected with B. abortus biotype 1, but not in cattle infected with B. melitensis biotype 3, sheep, or goats. The different results obtained with the serotype M and A polysaccharides and the sera from animals infected with M, A, and AM serotypes of Brucella spp. showed that in naturally infected animals, a large proportion of the antibodies are directed to or react with a previously defined common epitope(s) (J. T. Douglas and D. A. Palmer, J. Clin. Microbiol. 26:1353-1356, 1988) different from the A or M epitopes. By using the radial immunodiffusion test with B. melitensis 16M NH, it was possible to differentiate infected from vaccinated cattle, sheep, and goats with a sensitivity and specificity similar to that of the complement fixation test.     

Characterization of Brucella polysaccharide B.

Polysaccharide B was extracted from Brucella melitensis 16M and from a rough strain of Brucella abortus 45/20 by autoclaving or trichloroacetic acid extraction of whole cells and by a new method involving mild leaching of cells. The material obtained by either of the established procedures was contaminated by O polysaccharide. The new leaching protocol eliminated this impurity and provided a pure glucan, which was regarded as polysaccharide B. This polysaccharide was found by high-performance liquid chromatography separations, chemical composition, methylation, and two-dimensional homo- and heteronuclear magnetic resonance experiments to be a family of nonreducing cyclic 1,2-linked polymers of beta-D-glucopyranosyl residues. The degree of polymerization varied between 17 and 24. Polysaccharide B was essentially identical to cyclic D-glucans produced by Rhizobia, Agrobacteria, and other bacterial species. Pure polysaccharide B did not precipitate with Brucella anti-A or anti-M serum and did not inhibit the serological reaction of Brucella A or M antigen with either bovine or murine monoclonal Brucella anti-A or anti-M serum. Previously described serological reactions of polysaccharide B preparations with Brucella anti-A and anti-M sera are related in this study to the presence in crude extracts of contaminants with the antigenic properties of Brucella lipopolysaccharide O polysaccharides.     

Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge

znuA is known to be an important factor for survival and normal growth under low Zn(2+) concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (DeltaznuA) was constructed and found to be lethal in low-Zn(2+) medium. When used to infect macrophages, DeltaznuA B. abortus showed minimal growth. Further study with DeltaznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the DeltaznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.     

Cloning of a Brucella melitensis group 3 antigen gene encoding Omp28, a protein recognized by the humoral immune response during human brucellosis.

Brucella group 3 antigens (Ags) are outer membrane proteins (OMPs) with a molecular mass ranging from 25 to 30 kDa. The OMPs are of interest partially because of their potential use as vaccine and diagnostic reagents. We used human convalescent antibody (Ab) to clone a gene that encoded a 28-kDa protein from a lambdagt11 library of Brucella melitensis 16M genomic DNA. DNA sequence analysis revealed a single open reading frame that would encode a protein of 26,552 Da. The 28-kDa protein had a primary amino acid sequence that was 43% similar to a previously described Brucella abortus group 3 Ag, Omp25 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The similarity to a known group 3 OMP, immunoreactivity with Ab prepared against B. abortus group Ags, immunolabeling of whole cells, and Southern hybridization led to our conclusion that the B. melitensis 28-kDa protein was a group 3 protein distinct from B. abortus Omp25. We designated the B. melitensis protein Omp28. Human convalescent sera from patients infected with B. abortus and Brucella suis as well as rabbit antisera prepared against killed B. abortus whole cells recognized B. melitensis Omp28 on Western blots (immunoblots). Furthermore, mice and goats infected with smooth strains of B. melitensis produced Abs against Omp28. Our results may begin to explain the variability in molecular weight seen in Brucella group Ags and point toward their possible use in vaccination against infection as well as diagnosis of the disease.     

Conservation of seven genes involved in the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp

Seven genes of the wb locus of Brucella melitensis 16M involved in the biosynthesis of the lipopolysaccharide O-side chain have been recently identified, i.e. wbkA, gmd, per, wzm, wzt, wbkB, and wbkC, coding, respectively, for proteins homologous to mannosyltransferase, GDP-mannose 4,6 dehydratase, perosamine synthetase, ABC-type transporter (integral membrane protein), ABC-type transporter (ATPase domain), a hypothetical protein of unknown function, and a putative formyl transferase. The seven genes have a G + C content lower (around 48%) than that typical of Brucella spp. (58%) and thus may have been acquired from a species other than Brucella. In the present study, we analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) the seven O-chain biosynthetic genes for polymorphism among Brucella spp. PCR-RFLP showed that the seven genes are highly conserved and occur even in the naturally rough species B. ovis and B. canis and also in rough strains of B. abortus and B. melitensis. Nevertheless, the few polymorphisms that were observed consisted of absence of additional restriction sites sometimes allowing differentiation at the species level (e.g. B. ovis) or at the biovar or strain level. There were no apparent deletions or insertions in the PCR-amplified genes in any of the Brucella strains studied. In conclusion, the seven O-chain biosynthetic genes studied appear to be highly conserved among Brucella spp. and thus may have been acquired before species differentiation. Some of the species- or biovar-specific markers detected could be used for molecular typing of brucellae in addition to those previously described.     

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

Brucella abortus 1119-3 O-chain polysaccharide to differentiate sera from B. abortus S-19-vaccinated and field-strain-infected cattle by agar gel immunodiffusion.

Purified Brucella abortus 1119-3 and Brucella melitensis 16M lipopolysaccharide O-chain polysaccharides were not precipitated in agar gel immunodiffusion by any of 24 sera from vaccinated cattle but were precipitated by 18 of 24 sera from infected cattle. This difference can be used to differentiate sera of cattle vaccinated with B. abortus S-19 from sera of some field-strain-infected cattle.     

Definition of Brucella A and M epitopes by monoclonal typing reagents and synthetic oligosaccharides.

The paradigm that Brucella A and M epitopes are simultaneously expressed on single cells and within one antigen molecule was reinvestigated by using polysaccharide-specific murine monoclonal antibodies. Monoclonal antibodies were generated to the M antigen of Brucella melitensis 16M. Chemically defined lipopolysaccharides and O polysaccharides from Brucella abortus 1119-3, B. melitensis 16M, and Yersinia enterocolitica O:9 were used to dissect the binding profiles of the B. melitensis antibodies and an additional set of antibodies available from a B. abortus fusion experiment. Binding specificities were rationalized in terms of prototype A- and M-antigen structures, an interpretation supported by competitive binding studies with O polysaccharides and synthetic oligosaccharide analogs of the A and M antigens. Three binding patterns were characterized. Antibodies specific for the A antigen required five contiguous alpha 1,2-linked 4,6-dideoxy-4-formamido-D-mannopyranosyl residues, while antibodies with equal affinities for A or M epitopes were effectively inhibited by alpha 1,2-linked tri- or tetrasaccharides. Specificity for the M epitope correlated with binding of a critical disaccharide element alpha-D-Rha4NFo(1----3)alpha-D-Rha4NFo bracketed by alpha 1,2-linked residues. The binding profiles of Brucella monoclonal antibodies were consistent with the concept of simultaneous expression of A and M epitopes within a single molecule. A epitopes were present in the M antigen, and the discovery of isolated alpha 1,3 linkages in the A antigen suggests that M epitopes occur in all A antigens. Three monoclonal antibodies are proposed as standard reagents for the detection and identification of Brucella A and M antigens.     

A rapid and sensitive method for the identification of Brucella species with a monoclonal antibody.

A coagglutination test using monoclonal antibody BmE10-5 with specificity for the M antigen of Brucella melitensis 16M has been developed for the rapid identification of the smooth Brucella species. All reference strains of several biovars of B. melitensis, B. abortus, B. suis and B. neotomae tested were positive in this assay. No significant differences in reaction intensity were observed in relation to the different distribution of the A and M antigens among the Brucella serovars analysed. Conversely, rough Brucella species, with the exception of B. abortus 45/20, were negative in the assay. Among the different organisms tested not belonging to the genus Brucella, serovar O9 of Yersinia enterocolitica was the only one that gave a weak positive reaction of coagglutination. Thus, in view of its rapidity, simplicity, specificity and low costs, this technique could be highly useful for rapid identification of smooth Brucella strains in diagnostic laboratories.     

Epitope mapping of the Brucella melitensis BP26 immunogenic protein: usefulness for diagnosis of sheep brucellosis

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others (O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis- or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.     

Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp.

Immunological cross-reactions between Brucella spp. and Ochrobactrum anthropi were investigated in animals and humans naturally infected by Brucella spp. and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B. melitensis and Brucella abortus infected). In the animals tested, O. anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to that observed with B. melitensis brucellin. O. anthropi cytosolic proteins also reacted in gel precipitation tests with antibodies in sera from Brucella natural hosts with a frequency similar to that observed with B. melitensis proteins, and absorption experiments and immunoblotting showed antibodies to both Brucella-specific proteins and proteins common to Brucella and O. anthropi. No antibodies to O. anthropi cytosolic proteins were detected in the sera of Brucella-free hosts. Immunoblotting with sera of Brucella-infected sheep and goats showed immunoglobulin G (IgG) to Brucella group 3 outer membrane proteins and to O. anthropi proteins of similar molecular weight. No IgG to the O-specific polysaccharide of O. anthropi lipopolysaccharide was detected in the sera of Brucella-infected hosts. The sera of sheep, goats, and rabbits infected with B. melitensis contained IgG to O. anthropi rough lipopolysaccharide and lipid A, and B. ovis and O. anthropi rough lipopolysaccharides showed equal reactivities with IgG in the sera of B. ovis-infected rams. The findings show that the immunoresponse of Brucella-infected hosts to protein antigens is not necessarily specific for brucellae and suggest that the presence of O. anthropi or some related bacteria explains the previously described reactivities to Brucella rough lipopolysaccharide and outer membrane proteins in healthy animals.     

Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis

The identification of Brucella can be a time-consuming and labor-intensive process that places personnel at risk for laboratory-acquired infection. Here, we describe a real-time PCR assay for confirmation of presumptive Brucella isolates. The assay was designed in a multiplex format that will allow the rapid identification of Brucella spp., B. abortus, and B. melitensis in a single test.     

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.     

Immunochemical characterization of rough Brucella lipopolysaccharides

Lipopolysaccharides (LPS) were extracted from rough strains of Brucella abortus and Brucella melitensis and from strains of the naturally occurring rough species Brucella ovis and Brucella canis. Brucella rough lipopolysaccharides (R-LPS) were readily distinguished from Brucella smooth lipopolysaccharides (S-LPS) and enterobacterial R-LPS, by their chemical, physical, and serological characteristics. B. ovis R-LPS was differentiated from B. abortus, B. melitensis, and B. canis R-LPS by its reaction of partial identity in immunodiffusion. Monospecific mouse sera against B. ovis R-LPS agglutinated only the homologous bacteria but not R cells of other species of Brucella. B. ovis R-LPS contained more 2-keto, 3-deoxyoctonate, and glucosamine as a percentage of dry weight than any other R-LPS tested. B. abortus R-LPS was identified by the absence of an unidentified sugar present in the other R-LPS molecules, and B. melitensis R-LPS could be differentiated from B. canis R-LPS by its higher content of fatty acids. In contrast to S-LPS, all of the R-LPS studied lacked quinovosamine. In electron micrographs, Brucella R-LPS had a granular appearance, in contrast to typical lamellar structures formed by Brucella S-LPS and Escherichia coli R-LPS.     

Fine structure of A and M antigens from Brucella biovars.

Brucella A and M epitopes were found on single O-polysaccharide chains of all biotype strains of this species. Lipopolysaccharides from the type and reference strains of five of the six Brucella species, B. abortus, B. melitensis, B. suis, B. canis, and B. neotomae, were extracted and purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in conjunction with silver staining and immunoblotting developed by monoclonal antibodies, showed bands characteristic of A, M, or mixed A and M antigens. The A antigen previously described as an exclusively alpha 1,2-linked homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranose was shown by 1H and 13C nuclear magnetic resonance spectroscopy to possess a fine structure consistent with the low-frequency occurrence of alpha 1, 3-linked 4,6-dideoxy-4-formamido-D-mannopyranose residues. This feature was previously attributed only to the M antigen, which is also a homopolymer of the same sugar. B. melitensis biotype 3 and B. suis biotype 4 lipopolysaccharides showed characteristics of mixed A and M antigens. Immunoabsorption of these O polysaccharides on a column of immobilized A-antigen-specific monoclonal antibody enriched polymer chains with A-antigen characteristics but did not eliminate M epitopes. Composite A- and M-antigen characteristics resulted from O polysaccharides in which the frequency of alpha 1,3 linkages, and hence, M-antigen characteristics, varied. All biotypes assigned as A+ M- expressed one or two alpha 1,3-linked residues per polysaccharide O chain. M antigens (M+ A-) also possessed a unique M epitope as well as a tetrasaccharide determinant common to A-antigen structures. B. canis and B. abortus 45/20, both rough strains, expressed low-molecular-weight A antigen.     

Partial protection against Brucella infection in mice by immunization with nonpathogenic alphaproteobacteria

Previous findings indicate that Brucella antigens and those from nonpathogenic alphaproteobacteria (NPAP) are cross-recognized by the immune system. We hypothesized that immunization with NPAP would protect mice from Brucella infection. Mice were immunized subcutaneously with heat-killed Ochrobactrum anthropi, Sinorhizobium meliloti, Mesorhizobium loti, Agrobacterium tumefaciens, or Brucella melitensis H38 (standard positive control) before intravenous challenge with Brucella abortus 2308. Cross-reacting serum antibodies against Brucella antigens were detected at the moment of challenge in all NPAP-immunized mice. Thirty days after B. abortus challenge, splenic CFU counts were significantly lower in mice immunized with O. anthropi, M. loti, and B. melitensis H38 than in the phosphate-buffered saline controls (protection levels were 0.80, 0.66, and 1.99 log units, respectively). In mice immunized intraperitoneally with cytosoluble extracts from NPAP or Brucella abortus, protection levels were 1.58 for the latter, 0.63 for O. anthropi, and 0.40 for M. loti. To test whether the use of live NPAP would increase protection further, mice were both immunized and challenged by the oral route. Immunization with NPAP induced a significant increase in serum immunoglobulin G (IgG), but not serum or fecal IgA, against Brucella antigens. After challenge, anti-Brucella IgA increased significantly in the sera and feces of mice orally immunized with O. anthropi. For all NPAP, protection levels were higher than those obtained with systemic immunizations but were lower than those obtained by oral immunization with heat-killed B. abortus. These results show that immunization with NPAP, especially O. anthropi, confers partial protection against Brucella challenge. However, such protection is lower than that conferred by immunization with whole Brucella or its cytosoluble fraction.